Stabilized composition of tetrazolium salts

ABSTRACT

The present invention provides stabilized compositions of tetrazolium salts for analytical purposes, wherein they contain 1 to 10 moles of a complex-forming acid which is soluble in polar solvents per mole of tetrazolium salt. 
     The present invention also provides a process for the detection of reducing materials, wherein one of the above stabilized compositions is added to a test batch.

This is a continuation application of parent application Ser. No.328,315, filed Dec. 7, 1981, now abandoned.

This invention relates to a new stabilized preparation of tetrazoliumsalts for analytical purposes.

Tetrazolium salts have long been known in analytical chemistry for thedetection of reducing materials and especially of reducednicotinamideadenine dinucleotide (NADH). the transference of hydrogen iscatalysed not only by enzymes, such as diaphorase, but also by5-methylphenazinium methylsulphate (PMS) or similar substances, deepcoloured formazanes thereby being formed which permit a very sensitivedetection of the reducing substances in visible light. Therefore,appropriate processes have been developed in this way to detect a seriesof substances which are important in analytical chemistry, via the NADHproduced as intermediate. However, a disadvantage of this reaction isthat the tetrazolium salts decompose relatively easily, especially insolution, and form coloured decomposition products. Even in the case ofstorage at low temperatures and with the exclusion of light, thestability of such products is limited (see H. U. Bergmeyer, Grundlagender enzymatischen Analyse, pub. Verlag Chemie, Weinheim, 1977, pp.91-95, with further references).

Consequently, the problem exists of finding stabilising agents whichreduce or remove the sensitivity of the tetrazolium salts tocomparatively high temperatures and to light, without influencing thesensitivity of the detection reactions.

Thus, according to the present invention, there are provided stabilizedcompositions of tetrazolium salts for analytical purposes, wherein theycontain 1 to 10 moles and preferably 1 to 2 moles of a complex-formingacid which is soluble in polar solvents per mole of tetrazolium salt.

Conventionally employed tetrazolium salts, such as3-(4',5'-dimethylthiazolyl-2)-2,4-diphenyltetrazolium bromide (MTT),2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazolium chloride (INT),2,2',5,5'-tetra-(p-nitrophenyl)-3,3'-(3-dimethoxy-4-diphenylene)-ditetrazoliumchloride (TNBT),2,2'-di-(p-nitrophenyl)-5,5'-diphenyl-3,3'-dimethoxy-4,4'-diphenylene)-ditetrazoliumchloride (NBT), 2,2'-p-diphenylene-3,3',5,5'-tetraphenylditetrazoliumchloride (neotetrazolium chloride) (NT) and 2,3,5-triphenyltetrazoliumchloride (TT) are usually commercially available as chlorides orbromides and have hitherto been used as such in tests. An assumptionthat these salts hydrolyse partly in solution and that the instabilityis in some way connected therewith could not be confirmed since anaddition of conventional acids does not remove the instability.Conventional strong acids, such as toluenesulphonic acid, oxalic acid ormalonic acid, proved to be ineffective.

Surprisingly, we have now found that in part less acidic butcomplex-forming acids, such as boric acid or organichydroxypolycarboxylic acids, for example citric acid or malic acid,possess a very considerable stabilising effect not only againsttemperature stressing of the composition but also against illumination.Solutions of such a composition can, for example, be stored for severaldays at ambient temperature and in daylight without changing noticeably,i.e. their usefulness for analytical purposes is not lost. Commerciallyavailable forms of composition, for example lyophilisates orimpregnations on to absorbent carriers (reagent strips) can, afterstabilization according to the present invention, from findings which wehave already made, be kept for at least a year at ambient temperaturewithout noticeable decomposition.

Since the stabilising agents employed according to the present inventiondo not disturb the previously known test systems in which tetrazoliumsalts are used as indicators, this stabilization can be used for allknown tests. Thus, for example, mention may be made of the detection oflactic acid with lactate dehydrogenase, alcohol with alcoholdehydrogenase, glycerol with glycerol dehydrogenase, glucose withglucose dehydrogenase, acetaldehyde with acetaldehyde dehydrogenase, aswell as further systems which can be coupled to the above system, forexample via the formation of hydrogen peroxide and its reaction withalcohol and catalase to give acetaldehyde. Furthermore, strong reducingagents, for example ascorbic acid, can, of course, also reducetetrazolium salts directly to give the formazane.

The compositions according to the present invention are usually added tothe test system in the form of a solution but, for comparatively longterm storage, it is recommended to use them in solid form, for exampleas lyophilisates, powders or tablets or impregnated on to absorbentcarriers. Such absorbent carriers can, at the same time, be attached toa short handle so that they can be used as mixing device in the mannerdescribed in Federal Republic of Germany Patent Specification No.2,301,999.

The following Examples are given for the purpose of illustrating thepresent invention:

EXAMPLE 1 Reagent paper with3-(4,5-dimethylthiazolyl-2-2,4-diphenyltetrazolium bromide (MTT)

Filter paper (Schleicher & Schull No. 598) is impregnated with asolution of the following composition:

MTT: 0.25 mole

citric acid: 0.50 mole

methanol: add 1000 ml.

and then dried. A lemon yellow material is obtained which, upon elutioninto water, gives a bright yellow solution with a pH of 2.2 and which,protected against light, is stable for at least one year at ambienttemperature.

If a comparison is made with reagent papers prepared in an analogousmanner but which contain other acids instead of citric acid in the samemole ratio by measurement in a remission spectrophotometer (Zeiss DMR21) between 400 and 700 nm., then with increasing period of stressing, amore or less marked coloration is to be observed in the range of from560 to 580 nm., which is set out in the following Table. Thedecomposition product is preponderantly formazane.

    ______________________________________                                        acid variant                                                                             pH after % remission at 578 nm                                     (1 mole acid per                                                                         elution             12 weeks                                                                             36 weeks                                0.5 mole MTT)                                                                            in water unstressed at 25° C.                                                                     at 25° C.                        ______________________________________                                        without acid                                                                             5.5      62         60     42                                      tartaric acid                                                                            2.1      95         80     52                                      salicylic acid                                                                           2.2      67         64     55                                      oxalic acid                                                                              1.5      85         78     40                                      boric acid 4.5      93         91     80                                      citric acid                                                                              2.2      93         90     83                                      ______________________________________                                    

EXAMPLE 2 Lactate dehydrogenase (LDH) colour test with MTT/diaphorase

On the lower end of a reagent strip of 6 mm. breadth and above 75 mm.length are fixed 3 separate zones with the dimensions 6×6 mm., one ofwhich contains 1.3 mg. MTT, together with 1.3 mg. citric acidimpregnated on to filter paper (Schleicher & Schull No. 3455) frommethanolic solution, another contains 4.8 mg. NAD impregnated on topolyamide/cellulose fleece (Binzer No. VS 532) from aqueous solution andthe third contains 0.3 U diaphorase impregnated on topolyamide/cellulose fleece (Binzer No. VS 532) from 0.05 molartris-citrate buffer (pH 7). Elution into 2 ml. of a 0.1 molar tris/HClsolution (pH 8.0) which contains 0.5% detergent and 40 mMol/liter oflactate gives a reagent solution with the following composition:

tris/HCl buffer: 0.1 mol/l.

detergent: 0.5%

lactate: 0.04 mol/l.

NAD: 1.5 mmol/l.

MTT: 1.5 mmol/l.

diaphorase: 130 U/l.

citrate: 6.0 mmol/l.

0.02 ml. of serum is pipetted into 2 ml. of this solution, well mixedand the mixture left to stand for 3 minutes at ambient temperature.

The LDH enzyme reaction is then measured by continuous recording orreading off of the extinction after definite periods of time, forexample every 60 seconds.

Reagent test strips stored for a year at ambient temperature with theexclusion of light and moisture show results which are identical tothose obtained with freshly prepared reagent strips. The analysis of thecomponent materials gave the following values, referred to one reagentstrip:

    ______________________________________                                        component              6 months  12 months                                    material   unstressed  at 25° C.                                                                        at 25° C.                             ______________________________________                                        NAD        4.8 mg.     4.6 mg.   4.0 mg.                                      diaphorase 0.3 U       0.21 U    0.18 U                                       MTT        1.3 mg.     1.3 mg.   1.2 mg.                                      formazane  0.0         0.0       0.0                                          ______________________________________                                    

A reagent strip prepared identically except for the addition of citratebuffer and citric acid shows the following analytical values. Even inthe case of the unstressed papers, the formazane formed from the MTTdisturbs the measurement. After stressing, a measurement is no longerpossible:

    ______________________________________                                        component                    6 months                                         material       unstressed    at 25° C.                                 ______________________________________                                        NAD            4.8 mg.       4.6 mg.                                          diaphorase     0.3 U         0.21 U                                           MTT            1.2           1.0                                              formazane      0.1           0.3                                              ______________________________________                                    

It will be understood that the specification and examples areillustrative but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

What is claimed is:
 1. Method for the detection of reducing materials bymeans of tetrazolium salts which method comprises adding a tetrazoliumsalt stabilized by the addition of 1 to 10 moles of a complex-formingacid selected from the group consisting of boric acid and an organichydroxypolycarboxylic acid which is soluble in polar solvents, per moleof tetrazolium salts, to a solution buffered to the reaction conditionsof the other reagents; subsequently adding the sample thereto andmeasuring the color change.
 2. Method as claimed in claim 1, furthercomprising adding diaphorase as an activator for the tetrazolium salts.3. Method as claimed in claim 1, further comprising adding PMS as anactivator for the tetrazolium salts.
 4. The method as claimed in claim 1wherein 1 to 2 moles of the complex-forming acid is used per mole oftetrazolium salt.
 5. The method as claimed in claim 1, wherein thecomplex-forming acid is boric acid.
 6. The method as claimed in claim 1,wherein said complex forming acid is an organic hydroxypolycarboxylicacid.
 7. The method as claimed in claim 1, wherein the complex formingacid is citric acid.
 8. The method as claimed in claim 1, wherein thetetrazolium salt is3-(4',5'-dimethylthiazolyl-2)-2,4-diphenyltetrazolium bromide.
 9. Themethod as claimed in claim 1, wherein the tetrazolium salt is2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride.
 10. Themethod as claimed in claim 1, wherein the tetrazolium salt is2,2'-di-(p-nitrophenyl)-5,5'-diphenyl-3,3'-(3,3'-dimethoxy-4,4'-diphenylene)-ditetrazoliumchloride.
 11. The method as claimed in claim 1, wherein the stabilizedcomposition is in the form of a solution.
 12. The method as claimed inclaim 1, wherein the stabilized composition is a lyophilizate.
 13. Themethod as claimed in claim 1, wherein the stabilized composition isimpregnated into an absorbent carrier.